The orphan nuclear receptor ROR?t has critical roles in immune system development, homeostasis, and responses to microbial pathogens. This transcription factor is expressed only in lymphoid lineage cells, including immature thymocytes, lymphoid tissue inducer (Lti) cells, and T helper cells that produce the inflammatory cytokine IL-17 (Th17 cells). ROR?t has essential functions in each of these cells. It is required for the survival of CD4+8+ thymocytes and, hence, for the selection of an appropriate T cell repertoire; it is essential for the differentiation of Lti cells that induce formation of lymph nodes and Peyer's patches in the fetus and of cryptopatches and isolated lymphoid follicles in the post-natal intestine; and it is required for the differentiation of Th17 cells from nave CD4+ T lymphocytes. Th17 cells have recently been shown to be major mediators of disease in several models for autoimmunity in the mouse, and there is accumulating evidence that they have similar roles in humans. We found the absence of ROR?t in T cells results in reduced IL-17 production in response to relevant cytokines and in the loss of most Th17 cells in the intestinal lamina propria, where they are normally most abundant. In addition, mice lacking ROR?t are refractory to the induction of several autoimmune diseases. These findings suggest that ROR?t may be an attractive target for pharmacological intervention in multiple inflammatory conditions. Our understanding of the mechanism of action of ROR?t is currently limited, and little is known about how its transcriptional activity is regulated. There is compelling evidence that ROR?t, like other nuclear receptors, is regulated by binding of a ligand. In preliminary studies, we have shown that mutations in the putative ligand binding domain abrogate the ability of ROR?t to induce transcriptional activity or to direct cytokine gene expression in T cells. We have found that ROR?t has transcriptional activity in Drosophila S2 cells, and have exploited this observation to perform a genome-wide RNA interference screen. This screen has been complemented by studies with overexpression or RNAi knockdown of genes involved in lipid biosynthetic pathways that have pointed us towards candidate ligands. Based on these findings and on other preliminary studies, we propose the following aims: (1) to further characterize biosynthetic pathways to pinpoint enzymes involved in generation of a ROR?t ligand, using both mammalian and insect cell-based systems; (2) to validate candidate ligands using both biochemical/biophysical (e.g., binding assays and co-crystal structures) and functional approaches (e.g. mouse and human Th17 cell polarization); and (3) to characterize other factors that influence ROR?t transcriptional activity, including mouse and human orthologs of genes identified in the Drosophila RNAi screen and molecules identified by co-immunoprecipitation of complexes and by interaction screening with appropriate libraries in yeast. Together, these studies will provide a better understanding of the mechanism of action of ROR?t and will facilitate new strategies for therapy in autoimmune diseases. Public Health Relevance: The transcription factor ROR?t is required for the differentiation of T helper (Th17) cells that orchestrate inflammatory responses in numerous diseases. The ability of ROR?t to direct expression of target genes, such as the pro-inflammatory cytokine IL-17, is dependent on its binding of a yet undefined ligand. Identification of the relevant ligand will hasten the development of new approaches for inhibiting ROR?t activity and for treating a large number of autoimmune diseases and cancers whose progression depends on inflammatory processes.